The cleavage of the peptide bond is a subject of ongoing interest, being one of the most common and most important procedures in biochemistry. However, the extreme stability of this bond, with half-life for spontaneous hydrolysis estimated as 350-600 years at neutral pH and room temperature (Radzicka, A., Wolfenden, R., J. Am. Chem. Soc. 1996, 110, 6105-6109) limits the range of reagents for efficient peptide or protein cleavage. Proteolytic enzymes are natural reagents that cleave peptide bonds with various degrees of specificity. However, only a few of them are used routinely in industrial or laboratory practice, due to such limitations as their narrow requirements for temperature and pH. Therefore, new agents that provide the selective cleavage of peptides and proteins have gained increasing importance. Potential applications are foreseen in protein engineering. They include the removal of functional domains from recombinant polypeptides in protein purification procedures, processing of precursors of active proteins, etc.
Developments in recombinant DNA technology have made it possible to express a wide range of cloned foreign genes in host organisms such as bacteria and yeast. The usage of suitable recombinant polypeptides for the expression of peptides or proteins of interest has become a common practice. Such carriers have several advantages. They may be selected to increase solubility of proteins of interest, to prevent their degradation in host cells or to simplify purification and detection procedures. However, it is often necessary to remove the added functional partner from the polypeptide of interest by means of specific peptide bond cleavage. This concerns for example pharmaceutical applications, where foreign polypeptide sequences can elicit immune response in patients, or structural investigations. A suitable method of peptide bond cleavage must be both specific and efficient and must not yield unwanted side products. In particular, such a method should not introduce such modifications in the polypeptide of interest, which would be difficult to remove. Furthermore, for pharmaceutical or other life sciences applications, such a method should not pose a threat of contamination of the product with pathogens.
One common approach to the issue of specific peptide bond cleavage is to use proteolytic enzymes. The most frequently used ones include Factor Xa (for example Nagai et al., EP0161937 and Grandi et al., EP0505921), enterokinase (for example LaVallie, EP0679189 and Ley et al., US2005158838), and thrombin (for example Gilbert et al., EP0666920). However, several serious inconveniences accompany enzymatic proteolysis. These include non-specific proteolytic attack on the polypeptide of interest; a need for extended incubations, which can cause denaturation or aggregation of the polypeptide of interest; incomplete cleavage, which reduces the yield and/or introduces heterogeneity to the purified polypeptide; the need for additional purification steps to separate it from the fusion partner, deactivate and remove protease, and exchange buffer or salt. Finally, proteolytic enzymes are often expensive, and thus not feasible for the large-scale use in pharmaceutical, clinical and biotechnological applications.
Another family of methods for specific peptide bond cleavage is based on protein splicing with inteins, naturally occurring internal sequences, which undergo an intramolecular rearrangement, through the formation and subsequent hydrolysis of an active (thio)ester. The latter step leads to an elimination of the intein and recombination of the neighbouring sequences (Hiera et al. J. Biol. Chem. 1990, 265, 6726-6733). Recently a number of mutant inteins have been designed that are able to promote only the first step of protein splicing (Muir, Annu. Rev. Biochem. 2003, 72, 249-289). In this approach, the polypeptide of interest is fused to a self-cleavable intein domain, which can be cleaved alternatively via an intermolecular trans(thio)esterification reaction with external thiols, such as dithiotreitol, β-mercaptoethanol or cysteine. This increasingly popular methodology has several drawbacks. The cleavage reaction requires the addition of thiols that modify the C-terminus of the polypeptide of interest. It is strictly dependent on the preservation of native intein conformation, which results in specific narrowed requirements for reaction conditions. Also the large sizes of intein moieties constitute a disadvantage because they can diminish solubility and purification efficiency (Belfort et al. U.S. Pat. No. 6,933,362).
A further family of specific peptide bond cleavage methods is based on chemical cleavage agents. These often require harsh reaction conditions. Even when added at a great excess over the substrate, they tend to cleave only with partial selectivity and low yield. Cyanogen bromide is one of principal chemical reagents for peptide bond hydrolysis. Although used commonly, it has several serious shortcomings. It is volatile and very toxic, is applied at a 100-fold excess over methionine residues, for which it is specific, requires 70% formic acid as solvent, and gives several unwanted side reactions. As a consequence of its specificity for single methionine residues, for proteins with additional methionines cyanogen bromide produces protein fragments that are no longer native because methionine residues in them undergo irreversible modifications (Dimarchi, EP0134070). Another way of chemical cleavage is to react a protein or peptide with hydroxylamine, which cleaves the bond between the Asn and Gly residues. Disadvantages of this approach include side reactions of hydroxylamine with other Asn and Gly residues in a protein or peptide, yielding hydroxamates (Wang, CN1371918). The cleavage of an Asn-Gly bond in a protein or peptide was disclosed (Palm, WO952815), by treating it with a compound of the general formula R1—(CH2)n—NH—(CH2)n—R2, wherein R1 denotes NH2 or OH, R2 denotes hydrogen, lower alkyl, NH2, OH or halogen, n denotes an integer from 1 to 3, and m denotes 0 or an integer from 1 to 3. This is a generalisation of the hydroxylamine cleavage, possessing similar limitations. A chemical method for peptide bond cleavage at tryptophan residues was also disclosed (Richiyaado, EP0288272), by treating a peptide or protein with trifluoroacetic acid, in the presence of sulfoxide and chloride ions. Hinman et al. (EP0339217) disclosed the cleavage of peptide bonds by nucleophilic tertiary organophosphines. Another method includes the peptide bond cleavage between a Lys and a Cys residue. In this reaction the cysteine residue is first cyanogenated, then the peptide is treated with weak alkali and the amino group of the lysine acts as a nucleophilic group attacking the carbonyl carbon on the peptide bond between Lys and the cyanocysteine residue and cleaving this bond (Iwakura et al., JP10045796). The use of the chemical cleavage methods outlined above will in most cases generate non-specific protein fragmentation, since tryptophan residues, and Asn-Gly or Lys-Cys pairs are frequently found in proteins. Another serious disadvantage of these methods is the use of toxic or harmful chemicals.
Various metal ions, including Cu2+, Zn2+, Pt2+, Pd2+, Ni2+, Cd2+, Fe2+, Fe3+, Ce3+, Eu3+ and Pr3+ were studied as agents for non-enzymatic peptide bond cleavage—uncomplexed or complexed with specific ligands; the latter are often called artificial metallopeptidases. Two general cleavage mechanisms can be employed. Redox reactions of metal ion chelates, were studied, often in the presence of H2O2/ascorbate (for example, see Rana et al., J. Am. Chem. Soc. 1990, 112, 2457-2458). According to Linder et al. (WO2005005458) an amino acid sequence, comprising at least two amino acids selected from the group comprising histidine, lysine, tryptophan, arginine, tyrosine, phenylalanine and cysteine is constructed at the predetermined cleavage site and proteins or peptides are allowed to react with free metal ions in the buffer. The buffer contains a reducing or oxidizing agent which enhances the cleavage. In fact, redox cleavage of oligo-His and oligo-His-X sequences by Cu2+ and Co2+ ions was demonstrated only by gel electrophoresis, without a further analysis of cleavage products, and therefore the accuracy of cleavage by this reaction remains to be demonstrated. As with other redox reactions of metal ions, multiple side-reactions of free radical character should be expected, for example side chain oxidations.
An alternative chemistry, based upon hydrolysis reactions, is employed in the majority of other approaches. Suh and Son (EP 1381392) disclosed a family of synthetic catalysts of general formula (R)(Z)n, in which n denotes an integer of 1 or more, R represents a material capable of selectively recognizing and binding a target protein and Z represents an active metal ion-ligand complex. The cleavage was proved for Cu2+ and Co3+ complexes, using myoglobin and avidin as protein substrates, but sequence specificity was low. This methodology requires R to be designed individually for each substrate and cleavage site, and the synthesis of appropriate catalysts is laborious.
Many studies were made on model systems such as activated amides (Sayre et al., Inorg. Chem. 1992, 31, 935-937 and references therein) and dipeptides or small peptides (Fujii et al., J. Biol. Inorg. Chem. 2002, 7, 843-851 and references therein, Kassai et al., Inorg. Chem. 2004, 43, 6130-6132 and references therein), but the hydrolytic cleavage of proteins was achieved only in several cases. Smith et al. showed that Cu2+ ions, and also Ni2+, Zn2+, Mn2+, Mg2+, Ca2+, Fe2+ ions were able to cleave (with various efficacies) the Lys226-Thr227 peptide bond in the hinge region of human IgG1 but did not propose a molecular mechanism (Smith et al., Int. J. Peptide Protein Res. 1996, 48, 48-55). The scission of myoglobin with low sequence specificity was proved for a series of Cu2+ compounds (Zhang et al., Inorg. Chem., 2003, 42, 492) which exhibited two adjacent cleavage sites. Another study on BSA, using a macrocyclic Cu2+ complex, demonstrated several preferred sites of cleavage, but no clear sequence specificity could be observed (Hegg and Burstyn, J. Am. Chem. Soc. 1995, 117, 70115-7016). Although in some cases the protein cleavage may be rapid, the sequence specificity of such reactions is still poor and hard to predict (de Oliveira et al., Inorg. Chem., 2005, 44, 921-929).
Yashiro et al. studied the mechanism of hydrolysis of peptides containing Ser residues, with a low sequence specificity, defined as -Xaa-Ser-, where Xaa denotes any amino acid. The mechanism of this reaction consists of an acyl shift, with a possible role for Zn2+, Cu2+, Ni2+, Pr3+ or Eu3+ added as free ions or complex species to polarize the peptide carbonyl group by coordination, and to promote the nucleophilic attack of an intramolecular OH group (Yashiro et al., Org. Biomol. Chem., 2003, 1, 629-632 and references therein).
A different mechanism was proposed for hydrolytic reactions mediated by Pt2+ and Pd2+ complexes. Kostic and Zhu (U.S. Pat. No. 5,352,771) disclosed complexes of Pt2+ and Pd2+ which selectively bind to sulfur atoms in side chains of residues such as methionine, cysteine and S-alkyl cysteine and promote the cleavage of peptide bonds adjacent to these sulfur-containing residues. The disclosed mechanism of this process assumes that either an internal transfer of a water molecule from the metal complex to the amide group or the attack of the exogenous water molecule derived from the aqueous reaction medium on the amide group is the crucial step of this reaction. Although these inventors proposed a wide variety of peptides and proteins which could be hydrolysed with this method (e.g. myoglobin, Zhu et al., J. Biol. Inorg. Chem., 1998, 3, 383-391), the low pH of the reaction, around 2, together with a low sequential specificity, limit the range of applications. A similar peptide bond cleavage mechanism between X and Z residues was also demonstrated for a Pd2+ complex and peptides of X-Z-His sequences (Milovic et al., J. Am. Chem. Soc. 2003, 125, 781-788 and references therein). This methodology is well suited for protein fragmentation for mass spectroscopy. Yet another mechanism was proposed speculatively by Zhu and Kostic (Inorg. Chim. Acta 2002, 339, 104-110) for the cleavage of Ser/Thr-His/Met sequences in human serum albumin (HSA) by Pd2+ complexes. It includes an acyl group shift with the formation of an intermediate ester with the alcoholic group present in the Ser/Thr side chain. This mechanism was cited for the cisplatin-mediated selective hydrolytic cleavage of acetic acid from the Ac-Ser-Met dipeptide, but not for the reaction of the Ac-Ser-His dipeptide (Manka et al., J. Inorg. Biochem. 2004, 98, 1947-1956).
Recently, Dutca et al. (Inorg. Chem., 2005, 44, 5141-5146) reported enhanced cleavage of Met-X peptide bonds by a Pt2+ complex under ultraviolet or microwave irradiation.
Humphreys (WO0032795) proposed the cleavage sites comprising the -DKTH-, -DRSH-, -EKSH- or -DKSH- sequences which are specifically cleaved by Cu2+ ions at temperatures above 50° C. However, the Cu2+ related hydrolysis was demonstrated previously to occur simply for Ser-His and Thr-His sequences under such conditions (Allen and Campbell, Int. J. Pept. Protein Res. 1996, 48, 265-273) and therefore multiple unspecific cleavages should be expected for this method.
Altogether then, the reactions of metal ions proposed previously and presented above share a disadvantage of low sequence specificity, based on one or two adjacent amino acid residues. Therefore they are suited better for protein fragmentation than for selective cleavage of dedicated sites that would occur without side reactions.
The Ac-TESHHK-NH2 hexapeptide was recently found to undergo a slow, spontaneous, sequence-specific hydrolysis in the presence of Ni2+ ions in a phosphate buffer, at pH 7.4 and 37° C. (Bal et al., Chem. Res. Toxicol., 1998, 11, 1014-1023). A Ni2+ complex of the C-terminal tetrapeptide amide SHHK-NH2 was found to be the product of this reaction, with a yield between 3% and 9% after 140 hours of incubation, depending on the concentration of Ni2+ ions. The cleavage occurred therefore between the Glu and Ser residues. Subsequent studies revealed that a peptide of 34 residues, comprising the above sequence, was cleaved with an identical sequence specificity by Ni2+ ions under analogous conditions, but ca. five times faster (Bal et al., Chem. Res. Toxicol., 2000, 13, 616-624). The same work demonstrated that Cu2+ ions hydrolysed this 34-peptide with the same specificity, but slower. The other two metal ions tested, Co2+ and Zn2+ were inactive. An analogous hydrolysis reaction was also seen for Ni2+ ions and histone H2A, which was the source of the hexapeptide and 34-peptide sequences. Further studies on Ala-substituted hexapeptide analogues of Ac-TESHHK-NH2 indicated that the reaction proceeded at alkaline pH, and allowed to identify the Ser residue and the C-terminal H is residue as the ones necessary for it to occur. The substitution of the Glu residue with Ala did not affect the reaction (Mylonas et al., J. Chem. Soc., Dalton Trans., 2002, 4296-4306). This published research indicated that peptides which could potentially be hydrolysed under alkaline conditions in the presence of metal ions, such as Ni2+, are represented by a general sequence P1SA1HP2, where P1 and P2 represent any peptide sequences, and A1 represents any amino acid residue.
The above description clearly demonstrates that there is a need for a method of selective cleavage of a peptide bond in a peptide or a protein, which would combine advantages of chemical agents, such as low cost and easy removal of the cleaving agent, with advantages normally associated with enzymatic reactions, such as high sequence specificity and reproducibility of cleavage and a lack of side reactions.
The present invention provides such a method, based on a novel molecular mechanism involving metal ions.